Targeting pancreatic cancer metabolic dependencies through glutamine antagonism.

Pancreatic ductal adenocarcinoma (PDAC) cells use glutamine (Gln) to support proliferation and redox balance. Early attempts to inhibit Gln metabolism using glutaminase inhibitors resulted in rapid metabolic reprogramming and therapeutic resistance. Here, we demonstrated that treating PDAC cells with a Gln antagonist, 6-diazo-5-oxo-L-norleucine (DON), led to a metabolic crisis in vitro. In addition, we observed a profound decrease in tumor growth in several in vivo models using sirpiglenastat (DRP-104), a pro-drug version of DON that was designed to circumvent DON-associated toxicity. We found that extracellular signal-regulated kinase (ERK) signaling is increased as a compensatory mechanism. Combinatorial treatment with DRP-104 and trametinib led to a significant increase in survival in a syngeneic model of PDAC. These proof-of-concept studies suggested that broadly targeting Gln metabolism could provide a therapeutic avenue for PDAC. The combination with an ERK signaling pathway inhibitor could further improve the therapeutic outcome.

Autophagy supports mitochondrial metabolism through the regulation of iron homeostasis in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) cells maintain a high level of autophagy, allowing them to thrive in an austere microenvironment. However, the processes through which autophagy promotes PDAC growth and survival are still not fully understood. Here, we show that autophagy inhibition in PDAC alters mitochondrial function by losing succinate dehydrogenase complex iron sulfur subunit B expression by limiting the availability of the labile iron pool. PDAC uses autophagy to maintain iron homeostasis, while other tumor types assessed require macropinocytosis, with autophagy being dispensable. We observed that cancer-associated fibroblasts can provide bioavailable iron to PDAC cells, promoting resistance to autophagy ablation. To overcome this cross-talk, we used a low-iron diet and demonstrated that this augmented the response to autophagy inhibition therapy in PDAC-bearing mice. Our work highlights a critical link between autophagy, iron metabolism, and mitochondrial function that may have implications for PDAC progression.

The polar oxy-metabolome reveals the 4-hydroxymandelate CoQ10 synthesis pathway.

Oxygen is critical for a multitude of metabolic processes that are essential for human life. Biological processes can be identified by treating cells with 18O2 or other isotopically labelled gases and systematically identifying biomolecules incorporating labeled atoms. Here we labelled cell lines of distinct tissue origins with 18O2 to identify the polar oxy-metabolome, defined as polar metabolites labelled with 18O under different physiological O2 tensions. The most highly 18O-labelled feature was 4-hydroxymandelate (4-HMA). We demonstrate that 4-HMA is produced by hydroxyphenylpyruvate dioxygenase-like (HPDL), a protein of previously unknown function in human cells. We identify 4-HMA as an intermediate involved in the biosynthesis of the coenzyme Q10 (CoQ10) headgroup in human cells. The connection of HPDL to CoQ10 biosynthesis provides crucial insights into the mechanisms underlying recently described neurological diseases related to HPDL deficiencies1-4 and cancers with HPDL overexpression5.

Spontaneous hydrolysis and spurious metabolic properties of α-ketoglutarate esters.

α-ketoglutarate (KG), also referred to as 2-oxoglutarate, is a key intermediate of cellular metabolism with pleiotropic functions. Cell-permeable esterified analogs are widely used to study how KG fuels bioenergetic and amino acid metabolism and DNA, RNA, and protein hydroxylation reactions, as cellular membranes are thought to be impermeable to KG. Here we show that esterified KG analogs rapidly hydrolyze in aqueous media, yielding KG that, in contrast to prevailing assumptions, imports into many cell lines. Esterified KG analogs exhibit spurious KG-independent effects on cellular metabolism, including extracellular acidification, arising from rapid hydrolysis and de-protonation of α-ketoesters, and significant analog-specific inhibitory effects on glycolysis or mitochondrial respiration. We observe that imported KG decarboxylates to succinate in the cytosol and contributes minimally to mitochondrial metabolism in many cell lines cultured in normal conditions. These findings demonstrate that nuclear and cytosolic KG-dependent reactions may derive KG from functionally distinct subcellular pools and sources.

Functional Genomics Identifies Metabolic Vulnerabilities in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer characterized by complex metabolic adaptations that promote survival in a severely hypoxic and nutrient-limited tumor microenvironment (TME). Modeling microenvironmental influences in cell culture has been challenging, and technical limitations have hampered the comprehensive study of tumor-specific metabolism in vivo. To systematically interrogate metabolic vulnerabilities in PDA, we employed parallel CRISPR-Cas9 screens using in vivo and in vitro systems. This work revealed striking overlap of in vivo metabolic dependencies with those in vitro. Moreover, we identified that intercellular nutrient sharing can mask dependencies in pooled screens, highlighting a limitation of this approach to study tumor metabolism. Furthermore, metabolic dependencies were similar between 2D and 3D culture, although 3D culture may better model vulnerabilities that influence certain oncogenic signaling pathways. Lastly, our work demonstrates the power of genetic screening approaches to define in vivo metabolic dependencies and pathways that may have therapeutic utility.

Neurons Release Serine to Support mRNA Translation in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) tumors have a nutrient-poor, desmoplastic, and highly innervated tumor microenvironment. Although neurons can release stimulatory factors to accelerate PDAC tumorigenesis, the metabolic contribution of peripheral axons has not been explored. We found that peripheral axons release serine (Ser) to support the growth of exogenous Ser (exSer)-dependent PDAC cells during Ser/Gly (glycine) deprivation. Ser deprivation resulted in ribosomal stalling on two of the six Ser codons, TCC and TCT, and allowed the selective translation and secretion of nerve growth factor (NGF) by PDAC cells to promote tumor innervation. Consistent with this, exSer-dependent PDAC tumors grew slower and displayed enhanced innervation in mice on a Ser/Gly-free diet. Blockade of compensatory neuronal innervation using LOXO-101, a Trk-NGF inhibitor, further decreased PDAC tumor growth. Our data indicate that axonal-cancer metabolic crosstalk is a critical adaptation to support PDAC growth in nutrient poor environments.

Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I.

Immune evasion is a major obstacle for cancer treatment. Common mechanisms of evasion include impaired antigen presentation caused by mutations or loss of heterozygosity of the major histocompatibility complex class I (MHC-I), which has been implicated in resistance to immune checkpoint blockade (ICB) therapy1-3. However, in pancreatic ductal adenocarcinoma (PDAC), which is resistant to most therapies including ICB4, mutations that cause loss of MHC-I are rarely found5 despite the frequent downregulation of MHC-I expression6-8. Here we show that, in PDAC, MHC-I molecules are selectively targeted for lysosomal degradation by an autophagy-dependent mechanism that involves the autophagy cargo receptor NBR1. PDAC cells display reduced expression of MHC-I at the cell surface and instead demonstrate predominant localization within autophagosomes and lysosomes. Notably, inhibition of autophagy restores surface levels of MHC-I and leads to improved antigen presentation, enhanced anti-tumour T cell responses and reduced tumour growth in syngeneic host mice. Accordingly, the anti-tumour effects of autophagy inhibition are reversed by depleting CD8+ T cells or reducing surface expression of MHC-I. Inhibition of autophagy, either genetically or pharmacologically with chloroquine, synergizes with dual ICB therapy (anti-PD1 and anti-CTLA4 antibodies), and leads to an enhanced anti-tumour immune response. Our findings demonstrate a role for enhanced autophagy or lysosome function in immune evasion by selective targeting of MHC-I molecules for degradation, and provide a rationale for the combination of autophagy inhibition and dual ICB therapy as a therapeutic strategy against PDAC.

Selective Alanine Transporter Utilization Creates a Targetable Metabolic Niche in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) evolves a complex microenvironment comprised of multiple cell types, including pancreatic stellate cells (PSC). Previous studies have demonstrated that stromal supply of alanine, lipids, and nucleotides supports the metabolism, growth, and therapeutic resistance of PDAC. Here we demonstrate that alanine cross-talk between PSCs and PDAC is orchestrated by the utilization of specific transporters. PSCs utilize SLC1A4 and other transporters to rapidly exchange and maintain environmental alanine concentrations. Moreover, PDAC cells upregulate SLC38A2 to supply their increased alanine demand. Cells lacking SLC38A2 fail to concentrate intracellular alanine and undergo a profound metabolic crisis resulting in markedly impaired tumor growth. Our results demonstrate that stromal-cancer metabolic niches can form through differential transporter expression, creating unique therapeutic opportunities to target metabolic demands of cancer. SIGNIFICANCE: This work identifies critical neutral amino acid transporters involved in channeling alanine between pancreatic stellate and PDAC cells. Targeting PDAC-specific alanine uptake results in a metabolic crisis impairing metabolism, proliferation, and tumor growth. PDAC cells specifically activate and require SLC38A2 to fuel their alanine demands that may be exploited therapeutically.This article is highlighted in the In This Issue feature, p. 890.

Compensatory metabolic networks in pancreatic cancers upon perturbation of glutamine metabolism.

Pancreatic ductal adenocarcinoma is a notoriously difficult-to-treat cancer and patients are in need of novel therapies. We have shown previously that these tumours have altered metabolic requirements, making them highly reliant on a number of adaptations including a non-canonical glutamine (Gln) metabolic pathway and that inhibition of downstream components of Gln metabolism leads to a decrease in tumour growth. Here we test whether recently developed inhibitors of glutaminase (GLS), which mediates an early step in Gln metabolism, represent a viable therapeutic strategy. We show that despite marked early effects on in vitro proliferation caused by GLS inhibition, pancreatic cancer cells have adaptive metabolic networks that sustain proliferation in vitro and in vivo. We use an integrated metabolomic and proteomic platform to understand this adaptive response and thereby design rational combinatorial approaches. We demonstrate that pancreatic cancer metabolism is adaptive and that targeting Gln metabolism in combination with these adaptive responses may yield clinical benefits for patients.

Intracerebral Distribution of the Oncometabolite d-2-Hydroxyglutarate in Mice Bearing Mutant Isocitrate Dehydrogenase Brain Tumors: Implications for Tumorigenesis.

The prevalence of mutant isocitrate dehydrogenase 1 (IDH1) brain tumors has generated significant efforts to understand the role of the mutated enzyme product d-2-hydroxyglutarate (D2HG), an oncometabolite, in tumorigenesis, as well as means to eliminate it. Glymphatic clearance was proposed as a pathway that could be manipulated to accelerate D2HG clearance and dictated the study design that consisted of two cohorts of mice bearing U87/mutant IDH1 intracerebral tumors that underwent two microdialysis - providing D2HG interstitial fluid concentrations - sampling periods of awake and asleep (activate glymphatic clearance) in a crossover manner. Glymphatic clearance was found not to have a significant effect on D2HG brain tumor interstitial fluid concentrations that were 126.9 ± 74.8 µM awake and 117.6 ± 98.6 µM asleep. These concentrations, although low relative to total brain tumor concentrations of 6.8 ± 3.6 mM, were considered sufficient to be transported by interstitial fluid and taken up into normal cells to cause deleterious effects. A model of D2HG CNS distribution supported this contention and was further supported by in vitro studies that showed D2HG could interfere with immune cell function. The study provides insight into the compartmental distribution of D2HG in the brain, wherein the interstitial fluid serves as a dynamic pathway for D2HG to enter normal cells and contribute to tumorigenesis.